【作者】 李强；

【导师】 温进坤；

【作者基本信息】 河北医科大学 ， 生物化学与分子生物学， 2020， 硕士

【摘要】 血管紧张素Ⅱ（angiotensin Ⅱ,AngⅡ）对哺乳动物血压的调节至关重要,但是长期的高于生理浓度的AngⅡ刺激会对血管壁中层的血管平滑肌细胞（vascular smooth muscle cell,VSMC）造成损伤已经被广泛证实。研究证明,AngⅡ能引起VSMC炎性损伤、氧化应激以及细胞器损伤,如何对抗AngⅡ引起细胞损伤以及如何清除受损的细胞器成为最近研究的热点。近几年人们对细胞自噬的研究尤为广泛。线粒体自噬是细胞清除受损线粒体维持稳态的重要方式。线粒体自噬过程中参与的信号分子十分复杂,除了经典的PINK1/Parkin/LC3途径外,越来越多的调节网络被发现,如NIX/BNIP3途径、FUNDC1通路。此外,研究发现,线粒体自噬发生时,线粒体和内质网两种细胞器相互作用增多,提示细胞器之间的接触交流可能与细胞器自噬的发生存在某种联系。越来越多的证据表明,线粒体相关内质网膜（mitochondria-associated ER membrane,MAM）或线粒体-内质网结构耦联有重要的生物学功能,包括Ca²⁺储存、脂质生物合成、参与线粒体分裂、诱导自噬。有研究报道,定位于MAM上的脂质转运蛋白参与自噬体的形成。类固醇敏感性调节蛋白（st AR）相关脂质转运结构域蛋白starD7是细胞内参与非囊泡性脂质转运的重要脂质转运蛋白,但是starD7是否定位于MAM以及它在MAM中发挥的功能尚不清楚。虽然已经证明,高血压患者细胞中的线粒体结构、功能和稳态发生改变,线粒体损伤不仅影响细胞呼吸、细胞内钙稳态和ATP合成,而且产生活性氧。但目前对于线粒体损伤如何影响线粒体与内质网之间的相互作用以及哪些蛋白质调节线粒体-内质网结构耦联及其与线粒体自噬的关系研究尚不明确。研究MAM组成蛋白在VSMC线粒体自噬中的作用和机制,可以进一步深化对高血压引发血管损伤的认识,对心血管病防治有重要意义。第一部分AngⅡ诱导血管平滑肌细胞线粒体自噬目的:研究AngⅡ对VSMC线粒体自噬的影响。方法:1.利用Mito SOX?红色荧光探针检测AngⅡ对VSMC线粒体产生活性氧的影响。2.利用ATP生物发光检测方法评估AngⅡ对VSMC线粒体功能的影响。3.Western blot、免疫双荧光染色检查AngⅡ对线粒体自噬标志蛋白表达的影响。结果:1. AngⅡ促进血管平滑肌细胞产生线粒体活性氧线粒体活性氧水平升高是引起线粒体氧化损伤的关键因素。Mito SOX?红色荧光探针染色结果显示,AngⅡ处理后的VSMC中线粒体Mito SOX?红色荧光强度明显高于对照组。提示AngⅡ刺激能明显诱导VSMC产生线粒体活性氧。2.AngⅡ损伤血管平滑肌细胞线粒体功能线粒体的主要功能是为细胞提供能量。细胞内ATP水平是评估线粒体功能的重要方式。用化学发光法检测细胞内ATP含量。结果显示,与对照组相比,AngⅡ处理后VSMC中ATP含量明显降低。提示AngⅡ处理后损伤VSMC线粒体功能。3.AngⅡ诱导血管平滑肌细胞线粒体自噬线粒体自噬是清除损伤线粒体的重要方式。我们用AngⅡ处理VSMC后发现,线粒体自噬指标PINK1蛋白表达水平较对照组明显升高;LC3-Ⅱ/LC3-I比值水平明显升高;免疫荧光结果显示,PINK1在VSMC中表达显著上调并与线粒体共定位。小结:AngⅡ诱导VSMC线粒体产生活性氧,损伤VSMC线粒体功能,最终诱导VSMC线粒体自噬。第二部分starD7通过调节线粒体-内质网结构的耦联参与AngⅡ诱导的血管平滑肌细胞线粒体自噬目的:探讨starD7在AngⅡ诱导VSMC线粒体自噬中的作用及机制。方法:1.利用三重免疫荧光染色观察AngⅡ对starD7亚细胞定位的影响。2.免疫荧光染色观察线粒体-内质网接触位点。3.超速离心法和免疫荧光检查starD7在线粒体-内质网结构耦联中的定位。4三重免疫荧光染色和免疫印迹分析以及RT-PCR检查AngⅡ对starD7表达及线粒体-内质网接触位点定位的影响。5.Western blot、免疫双荧光染色检测敲低starD7对VSMC线粒体自噬的影响。结果:1. AngⅡ促进starD7与线粒体和自噬体在血管平滑肌细胞中的共定位免疫荧光结果显示,与未处理组（control）相比,AngⅡ处理使starD7明显向线粒体聚集,并且与自噬体标记物LC3共定位增多。提示AngⅡ促进starD7与线粒体和自噬体的共定位。2. 在VSMC,AngⅡ增加线粒体-内质网接触位点利用Mito-Tracker红色荧光探针标记线粒体,KDEL标记内质网,激光共聚焦显微镜观察可见,AngⅡ刺激使线粒体-内质网接触位点明显增加。3. starD7定位于线粒体-内质网接触位点Percoll超速离心法对VSMC进行亚细胞器分离。结果显示,starD7主要存在于MAM;免疫荧光染色显示,starD7与线粒体相关内质网膜标志蛋白GRP78在VSMC中共定位。4. AngⅡ促进starD7集聚于线粒体-内质网接触位点我们利用starD7、KDEL和Mito-Tracker三重免疫荧光染色研究三者之间的关系。结果显示,AngⅡ刺激使线粒体-内质网接触位点上的starD7显著增加;q RT-PCR和免疫印迹分析结果显示,AngⅡ在m RNA水平和蛋白水平均能促进starD7表达。以上结果表明,AngⅡ既诱导starD7表达,又促使starD7向线粒体-内质网接触位点的聚集。5. starD7参与AngⅡ诱导的血管平滑肌细胞线粒体自噬免疫印迹结果显示,与转染si-NC的细胞相比,敲低starD7取消了AngⅡ对PINK1和LC3蛋白表达的上调;双重免疫荧光结果显示,starD7敲低明显抑制AngⅡ对LC3表达和线粒体定位的促进作用。小结:starD7定位于线粒体-内质网接触位点;AngⅡ促进starD7与线粒体和自噬体在VSMC中共定位;AngⅡ刺激诱导VSMC线粒体-内质网接触位点增加并促进starD7向线粒体-内质网接触位点聚集;starD7参与AngⅡ诱导的VSMC线粒体自噬。结论:1.AngⅡ诱导VSMC线粒体自噬。2.starD7通过调节线粒体-内质网结构耦联参与AngⅡ诱导的VSMC线粒体自噬。更多还原

【Abstract】 Angiotensin Ⅱ（AngⅡ）is essential for the regulation of blood pressure in mammals,but long-term stimulation of higher concentration of AngⅡ can cause damage to vascular smooth muscle cells in the media wall of blood vessels.It has been proved that AngⅡ can cause inflammation,oxidative stress and organelle damage of VSMCs.How to provent cell injuries caused by AngⅡ and how to remove damaged organelles have become the focus of recent research.In recent years,cell autophagy has been extensively studied.Mitophagy is an important way to remove the damaged mitochondria and maintain homeostasis.In addition to the classical PINK1/Parkin/LC3pathway,more and more regulatory networks have been found,such as NIX/BNIP3 pathway and FUNDC1 pathway.In addition,it has been demonstrated that during the occurrence of mitophagy,the interaction of mitochondria with endoplasmic reticulum（ER）,occurs through mitochondria-associated ER membrane（MAM）,suggesting that the contact and communication between these two organelles might be related to the occurrence of organelle autophagy.Increasing evidence suggests that MAM serves as an important cellular signaling platform associated with several important functions,including Ca²⁺storage,lipid biogenesis,mitochondrial division and induction of autophagy.In recent years,the some MAM-related proteins are identified in the MAM.It has been reported that some lipid transfer proteins located on MAM are involved in the formation of autophagosome.St AR-related lipid transfer protein 7（starD7）is an important lipid transfer protein participated in the non-vesicular lipid transport in cells,but whether the starD7 locating in MAM structure is involved in mitophagy is still unclear.PartⅠ AngⅡ induces mitophagy in VSMCsObjective:To test whether AngⅡ induces the mitophagy of VSMCs.Methods:1.Mito SOX?red fluorescent probe was used to detect the changes in mitochondrial ROS levels in AngⅡ-treated VSMC.2.Luciferase-based luminescence-detected ATP level was used to evaluate the effect of AngⅡ on VSMC mitochondrial functions.3.The effect of AngⅡ on VSMC mitophagy was detected by using Western blot and immunofluorescence staining to examine mitophagy-related markers.Results:1. AngⅡ significantly elevates the levels of mitochondrial ROS in VSMCMito SOX?red fluorescent probe staining showed that the red fluorescence intensity of Mito SOX?in AngⅡ-treated VSMC was significantly higher than that in the control group,suggesting that AngⅡ stimulation facilitates the production of mitochondrial ROS in VSMCs,which is a prerequisite for the oxidative damage of mitochondria.2.AngⅡ can induce mitochondrial dysfunction in VSMCsCompared with the control group,ATP content in VSMCs was significantly decreased after AngⅡ treatment,suggesting that AngⅡ induces mitochondrial dysfunction in VSMCs.3.AngⅡ promotes the mitophagy in VSMCsAfter treating VSMCs with AngⅡ,the mitophagy marker PINK1was significantly upregulated compared with the control group.The LC3-Ⅱ/LC3-I ratio was also significantly increased.The immunofluorescence staining showed that,in AngⅡ-treated VSMCs,PINK1 was mainly localiz ed on the mitochondria.Summary:AngⅡ causes oxidative damage to VSMC mitochondria,thus leading to mitochondrial dysfunction and mitophagy.Part Ⅱ starD7 participates in AngⅡ-induced VSMC mitophagythrough regulating mitochondria-ER contact sitesObjective:To elucidate whether and how starD7 participates in mitophagy of VSMCs induced by AngⅡ.Method:1.The subcellular localization of starD7 was observed by triple immunofluorescence staining in AngⅡ-treated VSMCs.2.Immunofluorescence staining was used to observe the effect of AngⅡ on mitochondria-ER contact sites.3.The fractionation by ultra centrifugation and immunofluorescence staining were used to detect whether starD7 is localized at the mitochondria-ER contact sites.4.The starD7 expression and subcellular localization in AngⅡ-treated VSMCs were analyzed by triple immunofluorescence,western blot analysis and RT-PCR.5.Western blot and immunofluorescence staining were used to detect AngⅡ-induced mitophagy in starD7-knocked down VSMCs.Results:1. AngⅡ stimulation facilitates the co-localization of starD7 with mitochondria and autophagosome in VSMCsImmunofluorescence staining showed that the co-localization of starD7 with mitochondria and LC3 was increased significantly in AngⅡ–treated VSMCs.2. AngⅡ treatment increased in mitochondria-ER contact sites in VSMCsMito-Tracker red fluorescent probe was used to label the mitochondria,and KDEL was used to label the endoplasmic reticulum.Confocal microscopy results showed that the mitochondria-ER contact sites were significantly increased after AngⅡ treatment.3. starD7 is localized on the mitochondria-associated ER membraneThe rsubcellular localization by ultra centrifugation showed that starD7 was present in the isolated mitochondria-associated ER membrane.Immunofluorescence staining showed that starD7 co-locatlized with mitochondria-associated ER membrane marker GRP78.4. AngⅡ promotes the accumulation of starD7 at mitochondria-ER contact sitesTriple immunofluorescence staining showed that AngⅡ significantly promoted the accumulation of starD7 at mitochondria-ER contact sites.Western blot analysis and RT-PCR showed that AngⅡ promoted starD7 expression at both m RNA and protein levels.5. starD7 is involved in AngⅡ-induced VSMC mitophagyAfter starD7 was knocked down with small interfering RNA,western blot showed that the expression of PINK1 and LC3 was significantly decreased.Immunofluorescence staining showed that the level of mitophagy significantly decreased in starD7-knocked down VSMCs.Summary:starD7 is localized to mitochondria-associated ER membrane.AngⅡ promotes the accumulation of starD7 at the mitochondria-ER contact sites.After starD7 was knocked down in VSMCs,the level of mitophagy induced by AngⅡ was significantly reduced.Conclusion:1.AngⅡ induces mitophagy in VSMCs.2.starD7 participates in the mitophagy of VSMCs induced by through regulating mitochondria-ER contact sites.更多还原